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Dysregulated inflammation within the central nervous system (CNS) contributes to neuropathology in infectious, autoimmune, and neurodegenerative disease. With the exception of microglia, major histocompatibility complex (MHC) proteins are virtually undetectable in the mature, healthy central nervous system (CNS). Neurons have generally been considered incapable of antigen presentation, and although interferon gamma (IFN-γ) can elicit neuronal MHC class I (MHC-I) expression and antigen presentationin vitro, it remains unclear whether similar responses occurin vivo. Here we directly injected IFN-γ into the ventral midbrain of mature mice and analyzed gene expression profiles of specific CNS cell types. We find that IFN-γ induces cellular proliferation and expression of MHC-II and associated genes only in microglia. However, IFN-γ upregulated MHC-I and associated mRNAs in ventral midbrain microglia, astrocytes, oligodendrocytes, and GABAergic, glutamatergic, and dopaminergic neurons. The core set of IFN-γ-induced genes and their response kinetics were conserved across neurons and glia, with a lower amplitude of expression in neurons. A diverse repertoire of genes was upregulated in glia, particularly microglia, while no neuron-specific responses to IFN-γ were observed. Using mutant mice to selectively delete the IFN-γ-binding domain of IFNGR1 in dopaminergic neurons, we demonstrate that dopaminergic neurons respond directly to IFN-γ. Our results suggest that most neurons are capable of responding directly to IFN-γ and upregulating MHC-I and related genesin vivo, but their expression amplitude and repertoire is limited compared to oligodendrocytes, astrocytes, and microglia.One-sentence summaryWe find that IFN-γ induces transcription of MHC class I antigen processing and presentation machinery in all major parenchymal cell types in the ventral midbrain; however, neuronal responses are low amplitude and limited to a small set of genes, MHC class II expression and cellular proliferation are restricted to microglia, and dopamine neuronal responses require cell autonomous expression of IFNGR1.

Dopamine neurotransmission in the striatum is central to many normal and disease functions. Ventral midbrain dopamine neurons exhibit ongoing tonic firing that produces low extrasynaptic levels of dopamine below the detection of conventional extrasynaptic cyclic voltammetry (∼10–20 nanomolar), with superimposed bursts that can saturate the dopamine uptake transporter and produce transient micromolar concentrations. The bursts are known to lead to marked presynaptic plasticity via multiple mechanisms, but analysis methods for these kinetic parameters are limited. To provide a deeper understanding of the mechanics of the modulation of dopamine neurotransmission by physiological, genetic, and pharmacological means, we present three computational models of dopamine release with different levels of spatiotemporal complexity to analyze in vivo fast-scan cyclic voltammetry recordings from the dorsal striatum of mice. The models accurately fit to cyclic voltammetry data and provide estimates of presynaptic dopamine facilitation/depression kinetics and dopamine transporter reuptake kinetics, and we used the models to analyze the role of synuclein proteins in neurotransmission. The models’ results support recent findings linking the presynaptic protein α-synuclein to the short-term facilitation and long-term depression of dopamine release, as well as reveal a new role for β-synuclein and/or γ-synuclein in the long-term regulation of dopamine reuptake.

Both, patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector – carbon fiber electrode (CFE) – is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

Dopamine neurotransmission in the striatum is central to many normal and disease functions. Ventral midbrain dopamine neurons exhibit ongoing tonic firing that produce low extrasynaptic levels of dopamine below the detection of extrasynaptic electrochemical recordings (∼10 – 20 nanomolar), with superimposed bursts that can saturate the dopamine uptake transporter and produce transient micromolar concentrations. The bursts have previously been shown to lead to presynaptic plasticity via multiple mechanisms, but analysis methods for these kinetic parameters are limited. To provide a deeper understanding of the mechanics of dopamine neurotransmission, we present three computational models of dopamine release with different levels of spatiotemporal complexity to analyze in vivo fast-scan cyclic voltammetry recordings from the dorsal striatum of mice. The models accurately fit to the cyclic voltammetry data and provide estimates of presynaptic dopamine facilitation/depression kinetics and dopamine transporter reuptake kinetics. We use the models to analyze the role of synuclein proteins in neurotransmission and quantify recent findings linking presynaptic protein α-synuclein to the short-term facilitation and long-term depression of dopamine release.

Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

Optical imaging of changes in membrane potential of living cells can be achieved by the means of fluorescent voltage sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer supported probes for chemical, non-genetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) (PEG) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring in the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with non-genetically targeted VSDs in native brain tissue.

Local translation within excitatory and inhibitory neurons is involved in neuronal development and synaptic plasticity. Despite the extensive dendritic and axonal arborizations of central monoaminergic neurons, the subcellular localization of protein synthesis is not well-characterized in these populations. Here, we investigated mRNA localization and translation in midbrain dopaminergic (mDA) neurons, cells with enormous axonal and dendritic projections, both of which exhibit stimulation-evoked dopamine (DA) release. Using highly-sensitive ribosome-bound RNA-sequencing and imaging approaches in mDA axons, we found no evidence for axonal mRNA localization or translation. In contrast, mDA neuronal dendritic projections into the substantia nigra reticulata (SNr) contain ribosomes and mRNAs encoding the major components of DA synthesis, release, and reuptake machinery. Surprisingly, we also observed dendritic localization of mRNAs encoding synaptic vesicle-related proteins, including those involved in vesicular exocytic fusion. Our results are consistent with a role for local translation in the regulation of DA release from dendrites, but not from axons. Our translatome data further defined a molecular signature of the sparse mDA neurons resident in the SNr, including enrichment of Atp2a3/SERCA3, an ER calcium pump previously undescribed in mDA neurons.

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation that requires energy, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, exposure to psychological stress increases serum circulating cell-free mtDNA (ccf-mtDNA) levels. Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibited a 2-3-fold increase in ccf-mtDNA, with no change in ccf-nuclear DNA levels, establishing the magnitude and specificity for ccf-mtDNA reactivity. In cell-based studies, we show that glucocorticoid signaling - a consequence of psychological stress in humans - is sufficient to induce mtDNA extrusion in a time frame consistent with stress-induced ccf-mtDNA increase. Collectively, these findings provide evidence that acute psychological stress induces ccf-mtDNA and implicate neuroendocrine signaling as a potential trigger for ccf-mtDNA release. Further controlled work is needed to confirm that observed increases in ccf-mtDNA result from stress exposure and to determine the functional significance of this effect.

The brain and behavior are under energetic constraints, limited by mitochondrial energy production capacity. However, the mitochondria-behavior relationship has not been systematically studied on a brain-wide scale. Here we examine the association between multiple features of mitochondrial respiratory chain capacity and stress-related behaviors in mice with diverse behavioral phenotypes. Miniaturized assays of mitochondrial respiratory chain enzyme activities and mitochondrial DNA (mtDNA) content were deployed on 571 samples across 17 brain regions, defining specific patterns of mito-behavior associations that vary across brain regions and behaviors. Furthermore, multi-slice network analysis applied to our brain-wide mitochondrial dataset identified three large-scale networks of brain regions with shared mitochondrial signatures. A major network composed of cortico-striatal regions exhibited the strongest mitochondria-behavior correlations, suggesting that this mito-based network is functionally significant. Mito-based networks also overlap with regional gene expression and structural connectivity, thereby providing convergent multimodal evidence of mitochondrial respiratory chain functional organization anchored in gene, brain and behavior.

SummaryThe impact of long-term opioid exposure on the embryonic brain is crucial to healthcare due to the surging number of pregnant mothers with an opioid dependency. Current studies on the neuronal effects are limited due to human brain inaccessibility and cross-species differences among animal models. Here, we report a model to assess cell-type specific responses to acute and chronic fentanyl treatment, as well as fentanyl withdrawal, using human induced pluripotent stem cell (hiPSC)-derived midbrain organoids. Single cell mRNA sequencing (25,510 single cells in total) results suggest that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters the pathways associated with synaptic activities and neuron projection. Acute fentanyl treatment, however, increases dopamine release but does not induce significant changes in gene expressions of cell lineage development. To date, our study is the first unbiased examination of midbrain transcriptomics with synthetic opioid treatment at the single cell level.

Background: Hair greying is a hallmark of aging generally believed to be irreversible and linked to psychological stress. Methods: Here, we develop an approach to profile hair pigmentation patterns (HPPs) along individual human hair shafts, producing quantifiable physical timescales of rapid greying transitions. Results: Using this method, we show white/grey hairs that naturally regain pigmentation across sex, ethnicities, ages, and body regions, thereby quantitatively defining the reversibility of greying in humans. Molecularly, grey hairs upregulate proteins related to energy metabolism, mitochondria, and antioxidant defenses. Combining HPP profiling and proteomics on single hairs, we also report hair greying and reversal that can occur in parallel with psychological stressors. To generalize these observations, we develop a computational simulation, which suggests a threshold-based mechanism for the temporary reversibility of greying. Conclusions: Overall, this new method to quantitatively map recent life history in HPPs provides an opportunity to longitudinally examine the influence of recent life exposures on human biology. Funding: This work was supported by the Wharton Fund and NIH grants GM119793, MH119336, and AG066828 (MP)., eLife digest Hair greying is a visible sign of aging that affects everyone. The loss of hair color is due to the loss of melanin, a pigment found in the skin, eyes and hair. Research in mice suggests stress may accelerate hair greying, but there is no definitive research on this in humans. This is because there are no research tools to precisely map stress and hair color over time. But, just like tree rings hold information about past decades, and rocks hold information about past centuries, hairs hold information about past months and years. Hair growth is an active process that happens under the skin inside hair follicles. It demands lots of energy, supplied by structures inside cells called mitochondria. While hairs are growing, cells receive chemical and electrical signals from inside the body, including stress hormones. It is possible that these exposures change proteins and other molecules laid down in the growing hair shaft. As the hair grows out of the scalp, it hardens, preserving these molecules into a stable form. This preservation is visible as patterns of pigmentation. Examining single-hairs and matching the patterns to life events could allow researchers to look back in time through a person’s biological history. Rosenberg et al. report a new way to digitize and measure small changes in color along single human hairs. This method revealed that some white hairs naturally regain their color, something that had not been reported in a cohort of healthy individuals before. Aligning the hair pigmentation patterns with recent reports of stress in the hair donors’ lives showed striking associations. When one donor reported an increase in stress, a hair lost its pigment. When the donor reported a reduction in stress, the same hair regained its pigment. Rosenberg et al. mapped hundreds of proteins inside the hairs to show that white hairs contained more proteins linked to mitochondria and energy use. This suggests that metabolism and mitochondria may play a role in hair greying. To explore these observations in more detail Rosenberg et al. developed a mathematical model that simulates the greying of a whole head of hair over a lifetime, an experiment impossible to do with living people. The model suggested that there might be a threshold for temporary greying; if hairs are about to go grey anyway, a stressful event might trigger that change earlier. And when the stressful event ends, if a hair is just above the threshold, then it could revert back to dark. The new method for measuring small changes in hair coloring opens up the possibility of using hair pigmentation patterns like tree rings. This could track the influence of past life events on human biology. In the future, monitoring hair pigmentation patterns could provide a way to trace the effectiveness of treatments aimed at reducing stress or slowing the aging process. Understanding how ‘old’ white hairs regain their ‘young’ pigmented state could also reveal new information about the malleability of human aging more generally.

Significance We report a long-sought& in vivo& physiological role for& α-synuclein (α-syn) in dopamine signaling. The results indicate that& α-syn is critical for activity-dependent dopamine plasticity, and that short repeated burst activity produces rapid presynaptic facilitation, while prolonged burst activity slowly depresses evoked dopamine release. We propose that the rapid facilitation is due to an enhanced fusion of synaptic vesicles at active zones during exocytosis while the depression is due to synaptic exhaustion. These results identify a& dynamic role of& α-syn, and are critical for defining& molecular mechanisms and therapeutic targets for various neurological disorders, where the firing properties of neurons are severely altered., α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role in the regulation of neurotransmission under physiologically relevant conditions has proven elusive. We report that, in vivo, α-synuclein is responsible for the facilitation of dopamine release triggered by action potential bursts separated by short intervals (seconds) and a depression of release with longer intervals between bursts (minutes). These forms of presynaptic plasticity appear to be independent of the presence of β- and γ-synucleins or effects on presynaptic calcium and are consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. These results indicate that the presynaptic effects of α-synuclein depend on specific patterns of neuronal activity.

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.

α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role on neurotransmission under physiologically-relevant conditions has proven elusive. We report that α-synuclein is responsible for a rapid facilitation of dopamine release during action potential bursts in vivo. This occurs in tandem with a far slower stimulus-dependent depression, appears to be independent of the presence of β- and γ-synucleins or effects on presynaptic calcium and is consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. The results indicate that the presynaptic effects of α-synuclein are dependent on specific patterns of neuronal activity.

SummaryChanges in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson’s disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes are unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.

RATIONALE: GLT-1 is the major glutamate transporter in the brain and is expressed predominantly in astrocytes but is also present in excitatory axon terminals. To understand the functional significance of GLT-1 expressed in neurons, we generated a conditional GLT-1 knockout mouse and inactivated GLT-1 in neurons using Cre-recombinase expressed under the synapsin 1 promoter, (synGLT-1 KO). OBJECTIVES: Abnormalities of glutamate homeostasis have been shown to affect hippocampal-related behaviors including learning and memory as well as responses to drugs of abuse. Here, we asked whether deletion of GLT-1 specifically from neurons would affect behaviors that assessed locomotor activity, cognitive function, sensorimotor gating, social interaction, as well as amphetamine-stimulated locomotor activity. METHODS/RESULTS: We found that the neuronal GLT-1 KO mice performed similarly to littermate controls in the behavioral tests we studied. Although performance in open field testing was normal, the acute locomotor response to amphetamine was significantly blunted in the synGLT-1 KO (40% of control). We found no change in amphetamine-stimulated extracellular dopamine in the medial shell of the nucleus accumbens, no change in electrically stimulated or amphetamine-induced dopamine release, and no change in dopamine tissue content. CONCLUSIONS: These results support the view that GLT-1 expression in neurons is required for amphetamine-induced behavioral activation, and suggest that this phenotype is not produced through a change in dopamine uptake or release. Although GLT-1 is highly expressed in neurons in the CA3 region of the hippocampus, the tests used in this study were not able to detect a behavioral phenotype referable to hippocampal dysfunction.

Homozygous mutations in the aromatic l-amino acid decarboxylase (AADC) gene result in a severe depletion of its namesake protein, triggering a debilitating and often fatal form of infantile Parkinsonism known as AADC deficiency. AADC deficient patients fail to produce normal levels of the monoamine neurotransmitters dopamine and serotonin, and suffer a multi-systemic disorder characterized by movement abnormalities, developmental delay and autonomic dysfunction; an absolute loss of dopamine is generally considered incompatible with life. There is no optimal treatment for AADC deficiency and few truly good models in which to investigate disease mechanisms or develop and refine therapeutic strategies. In this study, we introduced a relatively frequently reported but mildly pathogenic S250F missense mutation into the murine Aadc gene. We show that mutants homozygous for the mutation are viable and express a stable but minimally active form of the AADC protein. Although the low enzymatic activity of the protein resulted in only modestly reduced concentrations of brain dopamine, serotonin levels were markedly diminished, and this perturbed behavior as well as autonomic function in mutant mice. Still, we found no evidence of morphologic abnormalities of the dopaminergic cells in mutant brains. The striatum as well as substantia nigra appeared normal and no loss of dopamine expressing cells in the latter was detected. We conclude that even minute levels of active AADC are sufficient to allow for substantial amounts of dopamine to be produced in model mice harboring the S250F mutation. Such mutants represent a novel, mild model of human AADC deficiency.

SummaryThe biophysical and biochemical properties of membrane lipids can alter the conformation and function of membrane-spanning proteins, yet the specific, physiological consequence in vivo of changing the membrane milieu for a specific protein has been rarely investigated. Using various genetic approaches to eliminate expression of the membrane-associated protein Flotillin-1, we have found that the lipid environment of the dopamine transporter (DAT) is necessary for mice to respond to amphetamine but not cocaine, because the localization of DAT to cholesterol-rich membranes is required for a DAT conformation that is essential for reverse transport of dopamine. Furthermore, a conditional rather than constitutive loss-of-function approach was necessary to reveal this phenotype, indicating a broader role for membrane-protein interactions that are modulated by Flotillin-1. Taken together, these findings demonstrate how interaction of a transmembrane protein with its membrane environment can regulate distinct events in the vertebrate brain that give rise to specific behavioral outcomes.

Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive parkinsonism. Multiple types of stress decrease parkin protein levels, an effect that may be relevant to sporadic Parkinson's disease (PD), but the mechanism(s) involved in this loss remain largely unclear. We sought to elucidate these mechanisms using a PD-relevant stressor, L-DOPA, the precursor to dopamine, which forms reactive oxygen species (ROS) as well as toxic quinones via auto-oxidation. We find that L-DOPA causes parkin loss through both an oxidative stress-independent and an oxidative stress-dependent pathway. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin's interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, mitochondrial parkin activity and autoubiquitination as well as mitophagy are not required for such loss. During stress induced by the oxidative stressor hydrogen peroxide or the metabolic uncoupler CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD patients, we suggest that these changes may lead to the loss of parkin expression in PD.

α-Synuclein (α-Syn) is a small cytosolic protein associated with a range of cellular compartments, including synaptic vesicles, the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. In addition to its physiological role in regulating presynaptic function, the protein plays a central role in both sporadic and familial Parkinson’s disease (PD) via a gain-of-function mechanism. Because of this, several recent strategies propose to decrease α-Syn levels in PD patients. While these therapies may offer breakthroughs in PD management, the normal functions of α-Syn and potential side effects of its depletion require careful evaluation. Here, we review recent evidence on physiological and pathological roles of α-Syn in regulating activity-dependent signal transduction and gene expression pathways that play fundamental role in synaptic plasticity.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer’s disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Dopamine neurotransmission in the dorsal hippocampus is critical for a range of functions from spatial learning and synaptic plasticity to the deficits underlying psychiatric disorders such as attention-deficit hyperactivity disorder. The ventral tegmental area (VTA) is the presumed source of dopamine in the dorsal hippocampus. However, there is a surprising scarcity of VTA dopamine axons in the dorsal hippocampus despite the dense network of dopamine receptors. We have explored this apparent paradox using optogenetic, biochemical, and behavioral approaches and found that dopaminergic axons and subsequent dopamine release in the dorsal hippocampus originate from neurons of the locus coeruleus (LC). Photostimulation of LC axons produced an increase in dopamine release in the dorsal hippocampus as revealed by high-performance liquid chromatography. Furthermore, optogenetically induced release of dopamine from the LC into the dorsal hippocampus enhanced selective attention and spatial object recognition via the dopamine D1/D5 receptor. These results suggest that spatial learning and memory are energized by the release of dopamine in the dorsal hippocampus from noradrenergic neurons of the LC. The present findings are critical for identifying the neural circuits that enable proper attention selection and successful learning and memory.

Summary Parkinson's disease (PD) is characterized by the selective loss of dopamine neurons in the substantia nigra; however, the mechanism of neurodegeneration in PD remains unclear. A subset of familial PD is linked to mutations in PARK2 and PINK1, which lead to dysfunctional mitochondria-related proteins Parkin and PINK1, suggesting that pathways implicated in these monogenic forms could play a more general role in PD. We demonstrate that the identification of disease-related phenotypes in PD-patient-specific induced pluripotent stem cell (iPSC)-derived midbrain dopamine (mDA) neurons depends on the type of differentiation protocol utilized. In a floor-plate-based but not a neural-rosette-based directed differentiation strategy, iPSC-derived mDA neurons recapitulate PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. We propose that these form a pathogenic loop that contributes to disease. Our study illustrates the promise of iPSC technology for examining PD pathogenesis and identifying therapeutic targets., Highlights • Disease modeling study with patient (monogenic)-derived iPSC for Parkinson's disease • Disease phenotypes exhibited by PD iPSC-derived midbrain DA neurons involved • Mitochondria, α-synuclein, selective vulnerability, and neurotransmitter regulation • These phenotypes may interact synergistically throughout PD progression, Shim, Studer, and colleagues demonstrate that using a floor-plate-based differentiation strategy, Parkinson's disease (PD) patient iPSC-derived mDA neurons recapitulate several PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. The authors further propose that these phenotypes form a pathogenic loop contributing to disease.

Neurotransmission at dopaminergic synapses has been studied with techniques that provide high temporal resolution, but cannot resolve individual synapses. To elucidate the spatial dynamics and heterogeneity of individual dopamine boutons, we developed fluorescent false neurotransmitter 200 (FFN200), a vesicular monoamine transporter 2 (VMAT2) substrate that selectively traces monoamine exocytosis in both neuronal cell culture and brain tissue. By monitoring electrically evoked Ca(2+) transients with GCaMP3 and FFN200 release simultaneously, we found that only a small fraction of dopamine boutons that exhibited Ca(2+) influx engaged in exocytosis, a result confirmed with activity-dependent loading of the endocytic probe FM1-43. Thus, only a low fraction of striatal dopamine axonal sites with uptake-competent VMAT2 vesicles are capable of transmitter release. This is consistent with the presence of functionally 'silent' dopamine vesicle clusters and represents, to the best of our knowledge, the first report suggestive of presynaptically silent neuromodulatory synapses.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic β-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic β-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in β-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in β-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which β-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, β-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

Dopamine (DA) is the most important catecholamine in the brain, as it is the most abundant and the precursor of other neurotransmitters. Degeneration of nigrostriatal neurons of substantia nigra pars compacta in Parkinson's disease represents the best-studied link between DA neurotransmission and neuropathology. Catecholamines are reactive molecules that are handled through complex control and transport systems. Under normal conditions, small amounts of cytosolic DA are converted to neuromelanin in a stepwise process involving melanization of peptides and proteins. However, excessive cytosolic or extraneuronal DA can give rise to nonselective protein modifications. These reactions involve DA oxidation to quinone species and depend on the presence of redox-active transition metal ions such as iron and copper. Other oxidized DA metabolites likely participate in post-translational protein modification. Thus, protein-quinone modification is a heterogeneous process involving multiple DA-derived residues that produce structural and conformational changes of proteins and can lead to aggregation and inactivation of the modified proteins.

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, psychological stress triggers elevation in circulating cell- free mtDNA (ccf-mtDNA). Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibit a 2-3-fold increase in ccf-mtDNA, with no change in nuclear DNA levels, establishing the magnitude and specificity to ccf-mtDNA. In cell-based studies, we show that glucocorticoid signaling – a consequence of psychological stress in humans – is sufficient to induce mtDNA extrusion in a time frame consistent with human psychophysiology. Collectively, these findings provide the first evidence that psychological stress induces ccf-mtDNA and implicate glucocorticoid signaling as a trigger for ccf-mtDNA release. Further work is needed to examine the functional significance of psychological stress-induced ccf-mtDNA as a mitokine in humans.

Modulation of corticostriatal synaptic activity by dopamine is required for normal sensorimotor behaviors. After loss of nigrostriatal dopamine axons in Parkinson's disease, l-3,4-dihydroxyphenlalanine (l-DOPA) and dopamine D2-like receptor agonists are used as replacement therapy, although these drugs also trigger sensitized sensorimotor responses including dyskinesias and impulse control disorders. In mice, we lesioned dopamine projections to the left dorsal striatum and assayed unilateral sensorimotor deficits with the corridor test as well as presynaptic corticostriatal activity with the synaptic vesicle probe, FM1-43. Sham-lesioned mice acquired food equivalently on both sides, while D2 receptor activation filtered the less active corticostriatal terminals, a response that required coincident co-activation of mGlu-R5 metabotropic glutamate and CB1 endocannabinoid receptors. Lesioned mice did not acquire food from their right, but overused that side following treatment with l-DOPA. Synaptic filtering on the lesioned side was abolished by either l-DOPA or a D2 receptor agonist, but when combined with a CB1 receptor antagonist, l-DOPA or D2 agonists normalized both synaptic filtering and behavior. Thus, high-pass filtering of corticostriatal synapses by the coordinated activation of D2, mGlu-R5, and CB1 receptors is required for normal sensorimotor response to environmental cues.

1-Methyl-4-phenylpyridinium (MPP(+)), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP(+) exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DAcyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP(+) exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP(+) concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP(+) depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DAcyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP(+)-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DAcyt levels in MPP(+)-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP(+)-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP(+) on neuronal DA homeostasis and neurotoxicity.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, invitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes invivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Levodopa replacement therapy has long provided the most effective treatment for Parkinson's disease (PD). We review how this dopamine (DA) precursor enhances dopaminergic transmission by providing a greater sphere of neurotransmitter influence as a result of the confluence of increased quantal size and decreased DA reuptake, as well as loading DA as a false transmitter into surviving serotonin neuron synaptic vesicles. We further review literature on how presynaptic dysregulation of DA release after l-dopa might trigger dyskinesias in PD patients. © 2014 International Parkinson and Movement Disorder Society

Significance Ca 2+ homeostasis is indispensable for the well being of all living organisms. Ca 2+ homeostasis is disrupted by α-synuclein (α-syn), whose misfolding plays a major role in neurodegenerative diseases termed synucleinopathies, such as Parkinson disease. We report that α-syn can induce sustained and highly elevated levels of cytoplasmic Ca 2+ , thereby activating a calcineurin (CN) cascade that results in toxicity. CN is a highly conserved Ca 2+ –calmodulin (CaM)-dependent phosphatase critical for sensing Ca 2+ concentrations and transducing that information into cellular responses. Limiting, but not eliminating, the availability of CaM, CN and/or CN substrates directly with genetic or pharmacological tools shifts the α-syn–induced CN cascade to a protective mode. This has mechanistic implications for CN's activity and provides a therapeutic venue for the treatment of synucleinopathies.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects., Amphetamines are known to enhance extracellular dopamine levels, but the underlying mechanisms are unclear. Utilising a new pH biosensor for synaptic vesicles, the authors show that amphetamines diminish vesicle pH gradients, disrupting dopamine packaging and leading to increased neurotransmitter release.

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of β2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.

Neural circuits are formed and refined during childhood, including via critical changes in neuronal excitability. Here, we investigated the ontogeny of striatal intrinsic excitability. We found that dopamine neuro-transmission increases from the first to the third postnatal week in mice and precedes the reduction in spiny projection neuron (SPN) intrinsic excitability during the fourth postnatal week. In mice developmentally deficient for striatal dopamine, direct pathway D1-SPNs failed to undergo maturation of excitability past P18 and maintained hyperexcitability into adulthood. We found that the absence of D1-SPN maturation was due to altered phosphatidylinositol 4,5-biphosphate dynamics and a consequent lack of normal ontogenetic increases in Kir2 currents. Dopamine replacement corrected these deficits in SPN excitability when provided from birth or during a specific period of juvenile development (P18–P28), but not during adulthood. These results identify a sensitive period of dopamine-dependent striatal maturation, with implications for the pathophysiology and treatment of neurodevelopmental disorders.